1. Field of the Invention
This invention relates to a method for determining free fatty acids in blood serum, e.g., palmitic acid, myristic acid, rinoleic acid, rinolic acid, etc., using fatty acid activating enzymes. More specifically, it relates to a method for determining free fatty acids in blood serum using a series of enzymatic reactions in which an acyl coenzyme A synthesizing enzyme (acyl-Co A synthetase, E. C. 6.2.1.3) and two phosphate group converting enzymes, myokinase (E. C. 2.7.4.3) and pyruvate kinase (E. C. 2.7.1.40), are successively reacted to form pyruvic acid in proportion to the amount of the fatty acids.
2. Description of the Prior Art
The determination of free fatty acids in blood serum has heretofore been attempted by various methods, such as extracting the free fatty acids with a lipid solubilizing organic solvent, and neutralizing and titrating them with alkali, or converting the free fatty acids to copper salts by the action of copper nitrate and triethanolamine, extracting the copper salts with chloroform, and subjecting the extract to the action of a chelating agent such as diethyldithiocarbamate to permit coloration.
The former method, however, has the defect of unsatisfactory reproducibility due to various factors such as the complexity of operation ascribable to the use of organic solvents, the effects of the presence of organic acids, and technical errors ascribable to titration. The latter method also suffers from the defect that the operation is complicated because of the formation of copper salts and the use of organic solvents, and that there is an undesired or toxic effect of organic solvents particularly chloroform, on the human body which cannot be overlooked. Both of these methods are thus very difficult to standardize as clinical diagnostic tests because of the complexity of operation.